Articles

CRISPR/Cas-9: An Exciting Addition to Genomic Editing

March 1, 2014

Rarely has a new technology generated the level of enthusiasm and interest that is now associated with CRISPR/Cas-9, a simple and efficient genomic editing technique with the potential for advancing basic genetic research, gene therapy, and personalized medicine. Scientists across the world, biotech and drug companies, and university licensing officers are capitalizing on the research opportunities presented by this technology and are using this versatile system on a multitude of species.

We have done an in-depth review of sources publicly available as of this writing and discuss here: (1) the science behind this new technology, (2) several of the research groups responsible for the promising applications for this technology, and (3) the related and evolving intellectual property (IP) landscape.

CRISPR is a developing technology with a promising—and unpredictable—future. Accordingly, we will track CRISPR’s progress over the coming months and will provide periodic updates.

The History and Science Behind CRISPR/Cas-9

CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats, which refers to regularly repeating nucleotide sequences originally noticed in bacterial genomes. These repeating sequences were first reported in 1987 by a Japanese research team examining the E. coli iap gene. The team noted the presence of these repeating sequences, but, at that time, could not attach biological significance to such “unusual structure[s]” consisting of multiple nucleotide sequences arranged as direct repeats.1

Nearly a decade later, a group from the Universidad de Alicante, Spain recognized that these tandem repeating sequences were a common feature in microbial genomes and referred to them as TREPs.2 The characteristic feature of what is now called CRISPR thus became recognized as a series of palindromic DNA repeats of about 20-50 base pairs, separated by spacer sequences of roughly the same length.

It was not until 2005, however, that a precise function was reported for these DNA repeats. Several groups reported that the bacterial spacer sequences located between the repeating palindromic sequences were often consistent with nucleotide sequences found in phages (bacterial viruses), thus suggesting a role in bacterial innate immunity; i.e. defense.

Then, in 2007, a research team led by Rodolphe Barrangou at the food ingredient and industrial enzymes company Danisco confirmed this possibility by demonstrating that bacterial resistance to a given phage strain could be altered by modifying the bacterial spacer DNA. It was thus understood that the nucleotide sequences between the palindromic repeats (spacers) correspond to DNA sequences of phage to which bacteria had been previously exposed, and that such spacers serve as “memory” of prior infection by the phage.

But “memory” alone is not enough to protect bacteria from subsequent exposure to phage. Further research has demonstrated that, in a bacterial cell, exposure to a previously-encountered phage triggers transcription of the corresponding spacer sequence into a short guide CRISPR-RNA (crRNA). The CRISPR-associated (“Cas”) enzyme known as the Cas-9 nuclease is then guided to the target phage by a two-part RNA structure that includes this crRNA and bears a short region of homology to the original phage DNA. This enables Cas-9 to identify and cleave the target phage DNA, effecting a double-stranded DNA cut at a specific site. It is this precise targeted DNA-cutting function of CRISPR/Cas9 systems that holds the most potential for use in genomic editing.

For genomic editing purposes, a researcher can simply mimic the capabilities of this two-part RNA structure by generating a single small “guide” RNA.3 This guide RNA can be programmed to correspond to any sequence of genomic DNA of interest and, when linked to a Cas-9 enzyme, constitutes an effective tool for the location and site-specific double-stranded cleavage of any target DNA.

CRISPR/Cas-9 Advantages

Researchers are now able to harness the site-specificity of the CRISPR/Cas9 for genomic editing purposes. Two important advantages of the CRISPR/Cas-9 system are its remarkable versatility working in cells and directly in eggs of multiple species (e.g. primate to malaria parasite to wheat) and its ease of use, including the cheapness of production.

In addition to its use for DNA cutting, the CRISPR/Cas-9 system can be used as a DNA nickase (a version of the Cas9 enzyme which generates a single-strand DNA break [nick], instead of a double-strand DNA break), can be used to run DNA interference with a catalytically inactive Cas-9 protein, and can be used to increase the expression of a gene by fusing Cas-9 to an activator protein. Through these techniques, CRISPR/Cas-9 can act as an RNA-guided platform for sequence-specific control of gene expression.4 Such gene expression control may prove particularly useful in drug design.

In contrast with existing genomic editing tools such as Zinc Finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs), the CRISPR/Cas-9 sgRNA editing system does not rely on protein design. This simplicity means that use of CRISPR/Cas-9 for genomic editing research requires considerably less time and fewer resources than ZFNs or even TALENs.

The Researchers

Scientists at several institutions have been working hard to understand the bacterial system and CRISPR and, particularly, to improve CRISPR’s functionality and harness its capabilities for ever-expanding research and biomedical applications.

Our research of publicly-available information showed there has been interest in CRISPR since at least 2007. In the following sections, we summarize work by those groups, including published papers and publicly-available patent filings. Each of these is discussed below, based on the chronological order of publications we identified.

University of Georgia: Rebecca Terns and Michael Terns of the University of Georgia have studied the roles of Cas proteins in the site-specific gene targeting mechanism of CRISPR. In 2008, they published their findings indicating that the Cas-6 protein functions in the generation of CRISPR guide-RNAs by cleaving longer RNA transcripts from the CRISPR loci.5

Northwestern University: Luciano Marraffini and Erik Sontheimer of Northwestern University published in 2008 on the application of CRISPR loci interference to counteract horizontal gene transfer and to limit the spread of antibiotic resistance in pathogenic bacteria such as Staphylococcus epidermidis.6

Danisco: In 2009, Danisco began exploiting CRISPR systems to improve the innate immunity of the bacterial cultures for yogurt and cheese manufacture.

University of California, Berkeley: In August of 2012, Jennifer Doudna, Emmanuelle Charpentier, Martin Jinek, and colleagues appear to be the first to publish their finding that the bacterial immunity system discovered by the Danisco group could be harnessed for genomic editing capabilities.7

Vilnius University, Lithuania: Less than one month later, in September of 2012, Giedrius Gasiunas, Rodolphe Barrangou, and colleagues also published on their understanding of the CRISPR/Cas-9 mechanism as paving the way for the development of unique molecular tools for RNA-directed DNA editing.8

The Broad Institute: Feng Zhang and colleagues at The Broad Institute published in early 2013 on the ability of CRISPR to enable simultaneous editing of several sites within the mammalian genome by using multiple guide RNA sequences.9

Harvard Medical School, Department of Genetics: Simultaneously, George Church and colleagues at Harvard Medical School, along with James DiCarlo of Boston University, reported on the capability to effect multiplex editing of target gene loci utilizing the introduction of multiple programmed guide RNAs. This group also reported, based on bioinformatics computation, that approximately 190,000 unique guide RNAs, which target approximately 40.5% of human exons, are available as potential target sites for interested researchers.10

The Whitehead Institute for Biomedical Research: Rudolph Jaenisch and colleagues at the Whitehead Institute, with Feng Zhang of The Broad Institute, published in May of 2013 on use of the CRISPR/Cas system to accomplish one-step generation of mice carrying mutations in multiple genes.11

On November 25, 2013, Editas Medicine announced it had received $43 million series A financing. The five founders are Feng Zhang, George Church, Jennifer Doudna, Keith Joung, and David Liu. Editas’ mission is described as translation of its “genome editing technology into a novel class of human therapeutics.”12

On January 23, 2014, Keith Joung and colleagues found that the possible undesirable side effect of CRISPR, i.e. off-target damage, could be reduced by >5000 fold by reducing the length of the guide targeting sequence from the traditional 20 bases to ~17-18 bases.13

The IP Landscape

Like the technology itself, the intellectual property landscape relating to CRISPR is evolving. As we carried out our research, it became increasingly clear that, because CRISPR is such a new technology, many of the patent filings relating to CRISPR as a tool for gene editing are only just now beginning to publish.14 As of the writing of this article, there are only a few published applications relating directly to CRISPR/Cas-9 as a genomic editing platform. Additionally, however, the CRISPR IP landscape includes several published applications and issued patents which are not directed specifically to genomic editing, but may be cited as prior art15 to later-filed pending patent applications. We summarize both aspects of this IP landscape below.

CRISPR-Related IP As Possible Prior Art

The Danisco team, which published in 2007 on the functional link between spacer sequences and bacterial immunity, filed several patent applications and has issued patents relating to CRISPR and bacterial typing, bacterial modifications, and methods for enhancing bacterial resistance to phage. For example, U.S. Patent Nos. 8,361,725 and 7,919,277 claim methods of typing a particular bacterial species having a CRISPR region by amplifying and typing the nucleotide sequences comprising that region.

Published applications pending from the Danisco group include U.S. App. No. 13/722,539, which presently claims an isolated nucleic acid molecule selected from several different CRISPR-associated sequence ID numbers, and U.S. App. No. 11/990,885, which presently claims a method for modulating resistance in a cell against a target nucleic acid comprising introducing one or more Cas genes or proteins into the cell in combination with a CRISPR sequence corresponding to the target nucleic acid.

The prosecution histories for these published applications are informative regarding the types of issues applicants are likely to face in attempting to describe and craft claims to protect this very new technology. For example, enablement and written description rejections have been made in several of these cases, with examiners indicating that broad claims to “CRISPR” encompass many possible sequences and are too broad.

There is also CRISPR work by Dr. Erik Sontheimer and colleagues at Northwestern University. Northwestern filed an application on September 23, 2009 (U.S. App. No. 12/565,589) directed to “Target DNA Interference with CrRNA.” This application published on March 25, 2010, before many of the filings discussed below.

Dr. Sontheimer’s initial claims were broadly directed to a “method of inhibiting the function and/or presence of a target DNA sequence” comprising administration of crRNA and one or more Cas proteins to a cell. The crRNA hybridizes with the target DNA, interfering with the function of a target sequence. The claims pursued are more narrowly directed to utilizing CRISPR for treating or preventing infections.

These claims were abandoned, and there are no pending U.S. applications in that family. During prosecution, the claims were rejected for inadequate enablement and lack of sufficient written description. The early-filed published application is available for all it teaches and may well be cited against later-filed applications drawn more specifically to claiming the use of CRISPR systems for genomic editing.

The University of Georgia Research Foundation (UGRF) secured a patent, U.S. Patent No. 8,546,553, on October 1, 2013 for “Prokaryotic RNAi-like System and Methods of Use,” by Terns et al. This patent includes claims directed to an isolated polynucleotide comprising at least 23 nucleotides, a psiRNA-tag (psi standing for “prokaryotic silencing”) and a guide sequence.

The focus of the invention described in this patent is modification of prokaryotic gene sequences. The claims seem limited only in that the CRISPR sequences used for such modifications are found in a microbe that employs the CRISPR system for immunity purposes

UGRF also has a published patent application (U.S. App. No. 13/127,764), which includes claims directed to Cas6 polypeptides and methods of use. This technology is listed on the University’s website as available for licensing.16 According to the website, “University of Georgia researchers have identified a novel RNA restriction endonuclease. Cas6 plays an essential role in the pRNAi pathway, cleaving CRISPR locus transcripts to generate individual invader-targeting RNAs.”

The present CRIPSR IP landscape includes PCT17 application WO 2013/176722, filed by the Regents of the University of California in conjunction with the University of Vienna and Jennifer Doudna. This PCT application names Martin Jinik, Emmanuelle Chapentier, Jennifer Doudna and others as inventors. It was filed on March 15, 2013, published on November 28, 2013, claimed priority back to May 25, 2012, and is entitled “Methods and Compositions for RNA-Directed Target DNA Modification and for RNA-Directed Modulation of Transcription.” It is an international application directed specifically to the use of the CRISPR/Cas-9 system for genomic editing (site-specific modification of a target DNA) purposes.

Several additional, recently-published PCTs describe more specific applications for the CRISPR/Cas-9 system, such as in therapeutics and more specific genomic editing strategies.

For example, on January 16, 2014, two PCTs from Sangamo Biosciences, Inc. published which describe the use of CRISPR/Cas-9 and other genomic editing platforms for specific therapeutic applications. One of these applications (PCT/US2013/032381) entitled “Methods and Compositions for the Treatment of Lysosomal Storage Diseases,” describes the delivery of a transgene into a cell using nucleases (such as ZFNs, TALENs or CRISPR/Cas-9 systems), such transgene thus enabling the cell to produce a protein capable of treating monogenic diseases such as Lysosomal storage disease.

Most recently, on January 30, 2014, an application published that is owned by The Broad Institute and MIT and names Feng Zhang among several inventors. This application (PCT/US2013/051418) describes, and presently claims, non-naturally occurring or engineered TALE or CRISPR-Cas systems comprising an inducible switch for the control (activation, enhancement, termination or repression) of specific genetic expression. This appears to be the MIT/Broad Institute’s first published application relating to their genomic editing work utilizing the CRISPR/Cas-9 system.

We expect the publication of additional applications relating to the CRISPR/Cas-9 system as a genomic editing tool or for specific therapeutic strategies, and we will monitor such developments.

A CRISPR International IP Snapshot

We have also surveyed published PCT applications relating to the use of CRISPR/Cas-9 for genomic editing or therapeutic applications. Although a few of the most recent of these are described in more detail in the text above, we provide the following table as a summary of each of these international applications that relate to the use of CRISPR/Cas-9 for genomic editing or therapeutic application.

Conclusion

Although the technological capabilities of CRISPR/Cas-9 for genomic editing are rapidly advancing, there are still many unanswered questions about how this technology will be available to the biomedical research community from an IP perspective. Many believe that CRISPR/Cas-9 will prove itself as the platform for the next great leap forward in genetic therapeutics and personalized medicine. However, no one can predict whether (or by whom) the technology will be controlled.

Our best advice is to stay tuned. Further developments are likely just around the corner as more patent applications publish and are granted, although it may be years before the patent landscape is completely defined.

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3 Jinek, M. et al., “A Programmable Dual-RNA—Guided DNA Endonuclease in Adaptive Bacterial Immunity,” Science 337, 816-821 (2012).
4 Qi, L.S. et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression,” Cell 152, 1173-1183 (2013).
5 Carte, J. et al., “Cas6 is an Endoribonuclease that Generates Guide RNAs for Invader Defense in Prokaryotes,” Genes & Development 22, 3489-3496 (2008).
6 Marraffini L.A. et al., “CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA,” Science 322, 1843-1845 (2008).
7 Jinek, M. et al., “A Programmable Dual-RNA—Guided DNA Endonuclease in Adaptive Bacterial Immunity,” Science 337, 816-821 (2012).
8 Gasiunas, G. et al., “Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria,” PNAS 109, E2579-E2586 (2012).
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10 Mali, P. et al. “RNA-Guided Human Genome Engineering via Cas9,” Science 339, 823-826 (2013).
11 Wang, H. et al., “One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering,” Cell 153, 910-918 (2013).
12 Editas press release, November 25, 2013.
13 Fu, Y. et al, “Improving CRISPR-Cas nuclease specificity using truncated guide RNAs,” Nature Biotechnology advance online publication, January 26, 2014.
14 A patent application does not become public upon filing. With some limited exceptions, such as request by the applicant for an earlier publication date, an application for a patent is published after the expiration of a period of 18 months from the earliest filing date for which a benefit is sought.
15 The term “prior art” refers to information that has been made publicly available in any form before a given date that might be relevant to a patent's claims of originality. If an invention has been described in the prior art, a patent on that invention is not valid.
16http://www.ovpr.uga.edu/technologies/detail/ncd-1492/
17 The Patent Cooperation Treaty (PCT) is an international patent law treaty that provides a unified procedure for filing patent applications to protect inventions in each of its contracting states. A patent application filed under the PCT is called an international application, or a PCT application.